Mammarenaviruses are significant human pathogens for which FDA-approved vaccines or treatments do not exist. While these viruses cause severe disease in humans, they are completely asymptomatic in their rodent hosts, where they establish a persistent, life-long infection. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) is carried by the common house mouse in nature and is transmitted vertically from mother to pup. The pups are born infected but never mount an effective immune response to clear the virus as viral proteins are seen as self-antigens by the pup?s developing immune system. Paradoxically, while LCMV can infect most cells in the host rodent, it tightly regulates its spread and therefore does not overrun its host. A favored hypothesis for how LCMV restricts its spread is through the production of defective interfering (DI) particles, which interfere with the ability of standard infectious virus particles to successfully complete the viral life cycle. A single DI particle entering a permissive host cell is sufficient to render that cell refractory to subsequent infection by a standard infectious virus particle. Thus, a virus that produces DI particles can limit its rate of spread to shield its host from the negative effects of infection while still retaining its ability to propagate and maintain itself in nature. The mechanism by which arenavirus DI particles interfere with standard virus propagation is unknown. For many RNA viruses, defective genomes containing large deletions in ORFs and/or promotor regions have been shown to be the molecular basis for interference. However, despite efforts to fully sequence the LCMV genome in the 1990s, no such deletions were observed. Instead, small deletions in the terminal 3? and 5? untranslated regions of the LCMV genome were detected. However, it is unknown whether these genomes are packaged into DI particles or can interfere with standard virus replication. Further, additional candidate defective genomes likely exist. In this application, we propose to apply next-generation sequencing technologies to identify candidate DI genomes in the LCMV model and functionally test whether they are indeed the basis for DI particle-mediated interference. If successful, the proposed experiments will provide the first comprehensive map of LCMV genomes and identify the molecular signature of DI particles. Further, these studies will answer a seminal question in the field by determining whether defective genomes are in fact responsible for blocking standard virus propagation or whether an alternative mechanism is at work. This fundamental information is necessary for future studies to fully define the mechanisms of DI particle formation and function.